September 4th
The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.
| 9/3 GAL4 | 40uL |
| M buffer(TOYOBO) | 5uL |
| XbaⅠ(TOYOBO) | 0.5uL |
| SpeⅠ(TOYOBO) | 0.5uL |
| dH2O | 4uL |
| Total | 50uL |
| each DNA | 40uL |
| 3 buffer(NEB) | 5uL |
| BglⅡ(NEB) | 0.5uL |
| dH2O | 4.5uL |
| Total | 50uL |
| 9/3 GAL4 | 23uL |
| M buffer(TOYOBO) | 10uL |
| XbaⅠ(TOYOBO) | 1uL |
| SpeⅠ(TOYOBO) | 1uL |
| CIP | 0.5uL |
dH2O | 64.5uL |
Total | 100uL |
September 5th
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
| EGFP | pSB1C3 |
| DNA template | 40uL | 23uL |
| 3 buffer | 5uL | 5uL |
| BglⅡ | 0.5uL | 0.5uL |
| dH2O | 4.5uL | 21.5uL |
Total | 50uL | 50uL |
| DNA template | 40uL |
| 4 buffer(NEB) | 5uL |
| SpeⅠ | 0.5uL |
| 100×BSA | 0.5uL |
| CIP | 0.5uL |
dH2O | 4uL |
Total | 50uL |
| 1uL |
| 1uL |
| dH2O | 3uL |
| Ligation high | 2.5uL |
Total | 7.5uL |
September 6th
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
| All samples |
| DNA template | 0.25uL |
| 10× KOD plus buffer | 5uL |
| 2mM dNTPs | 5uL |
| 25mM MgSO4 | 1.6uL |
| 10P 5’ primer | 1.5uL |
| 10P 3’ primer | 1.5uL |
| KOD plus | 1uL |
| dH2O | 34.15uL |
| Total | 50uL |
| Temperature | Time | Cycle |
| 95℃ | 2min | |
| 95℃ | 15sec | 30 cycle |
| 58℃ | 30sec | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | |
| 14℃ | ∞ | |
| pSB1C3(XbaⅠ and SpeⅠ cut) | 1uL |
| GAL4(XbaⅠ and SpeⅠ cut) | 1uL |
| dH2O | 3uL |
| Ligation high | 2.5uL |
| Total | 7.5uL |
September 7th
The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.
| All samples |
| DNA template | 0.25uL |
| 10× KOD plus buffer | 5uL |
| 2mM dNTPs | 5uL |
| 25mM MgSO4 | 1.6uL |
| 10P 5’ primer | 1.5uL |
| 10P 3’ primer | 1.5uL |
| KOD plus | 1uL |
| dH2O | 34.15uL |
| Total | 50uL |
| Temperature | Time | Cycle |
| 95℃ | 2min | |
| 95℃ | 15sec | 30 cycle |
| 58℃ | 2min30sec | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | |
| 14℃ | ∞ | |
September 8th
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃.
| All samples |
| DNA template | 1uL |
| 10× KOD plus buffer | 5uL |
| 2mM dNTPs | 5uL |
| 25mM MgSO4 | 1.6uL |
| 10P 5’ primer | 1.5uL |
| 10P 3’ primer | 1.5uL |
| KOD plus | 1uL |
| dH2O | 33.4uL |
| Total | 50uL |
| Temperature | Time | Cycle |
| 95℃ | 2min | |
| 95℃ | 15sec | 30 cycle |
| 58℃ | 2min30sec | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | |
| 14℃ | ∞ | |
| All samples |
| DNA template | 1uL |
| 10× KOD plus buffer | 5uL |
| 2mM dNTPs | 5uL |
| 25mM MgSO4 | 1.6uL |
| 10P 5’ primer | 1.5uL |
| 10P 3’ primer | 1.5uL |
| KOD plus | 1uL |
| dH2O | 33.4uL |
| Total | 50uL |
| Temperature | Time | Cycle |
| 95℃ | 2min | |
| 95℃ | 15sec | 30 cycle |
| 55℃(-1) or 58℃(-2) | 2min30sec | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | 30 cycle |
| 68℃ | 30sec(UAS) or 2min10sec(Except for UAS) | |
| 14℃ | ∞ | |
September 9th
The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. In total 83 flies were eclosed from the microinjected embryos. The calculated viability for the microinjected flies was 12.0%. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
TNFAIP3
Drosophila S2 cells (2.5 X 10
5 cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.
| UAS | 40uL |
| 4 buffer(NEB) | 5uL |
| EcoRⅠ-HF(NEB) | 0.5uL |
| SpeⅠ(NEB | 0.5uL |
| 100×BSA | 0.5uL |
| dH2O | 3.5uL |
| Total | 50uL |
| All sample |
| DNA template | 1uL |
| 10× KOD plus buffer | 5uL |
| 2mM dNTPs | 5uL |
| 25mM MgSO4 | 1.6uL |
| 10P 5’ primer | 1.5uL |
| 10P 3’ primer | 1.5uL |
| KOD plus | 1uL |
| dH2O | 33.4uL |
| Total | 50uL |
| Temperature | Time | Cycle |
| 95℃ | 2min | |
| 95℃ | 15sec | 30 cycle |
| 58℃ | 2min30sec | 30 cycle |
| 68℃ | 2min10sec | 30 cycle |
| 68℃ | 2min10sec | |
| 14℃ | ∞ | |
| 1uL |
| 1uL |
| dH2O | 3uL |
| Ligation high | 2.5uL |
| Total | 7.5uL |
| 1uL |
| dH2O | 4uL |
| Ligation high | 2.5uL |
| Total | 7.5uL |
September 10th
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.
TNFAIP3
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.
| HS or Act5c | 40uL |
| 4 buffer(NEB) | 5uL |
| XbaⅠ-HF(NEB) | 0.5uL |
| BamHⅠ(NEB ) | 0.5uL |
| 100×BSA | 0.5uL |
| dH2O | 3.5uL |
| Total | 50uL |
| G-1 and G-2 | G-3 and G-4 |
| DNA template | 1uL | 4uL |
| 10× KOD plus buffer | 5uL | 5uL |
| 2mM dNTPs | 5uL | 5uL |
| 25mM MgSO4 | 1.6uL | 1.6uL |
| 10P 5’ primer | 1uL | 1uL |
| 10P 3’ primer | 1uL | 1uL |
| KOD plus | 1uL | 1uL |
| dH2O | 33.4uL | 31.4uL |
| Total | 50uL | 50uL |
| Temperature | Time | Cycle |
| 95℃ | 2min | |
| 95℃ | 15sec | 30 cycle |
| 57℃(G-1 and G-3) or 59℃(G-2 and G-4) | 2min30sec | 30 cycle |
| 68℃ | 2min10sec | 30 cycle |
| 68℃ | 2min10sec | |
| 14℃ | ∞ | |
| pSB1C3(PCR) | 40uL |
| 4 buffer(NEB) | 5uL |
| EcoRⅠ-HF(NEB) | 0.5uL |
| SpeⅠ(NEB) | 0.5uL |
| 2100×BSA | 0.5uL |
| dH2O | 3.5uL |
| Total | 50uL |
| 13uL |
| 3 buffer(NEB) | 2uL |
| BglⅡ(NEB) | 0.5uL |
| dH2O | 2.5uL |
| Total | 20uL |
| GAL4 | 20uL |
| 4 buffer(NEB) | 4uL |
| XbaⅠ(NEB) | 0.7uL |
| SpeⅠ(NEB) | 0.7uL |
| 100×BSA | 0.4uL |
| dH2O | 14.6uL |
| Total | 40uL |
| GAL4 | 40uL |
| 3 buffer(NEB) | 5uL |
| BglⅡ(NEB) | 0.5uL |
| dH2O | 4.5uL |
| Total | 50uL |
| GAL4(Bgl Ⅱ cut) | 40uL |
| 4 buffer(NEB) | 5uL |
| SpeⅠ | 0.5uL |
| 100×BSA | 0.5uL |
| dH2O | 4uL |
| Total | 50uL |
| 1uL |
| 2uL |
| EGFP or LacZ | 2uL |
| Ligation high | 5uL |
| Total | 10uL |
| 1uL |
| 2uL |
| 2uL |
| Ligation high | 5uL |
| Total | 10uL |
"
